Following the discovery of protein modification by the small, highly conserved ubiquitin polypeptide, a number of distinct ubiquitin-like proteins (Ubls) have been found to function as protein modifiers as well These Ubls, which include SUMO, ISG15, Nedd8, and Atg8, function as critical regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy, and cell-cycle control A growing body of data also implicates the dysregulation of Ubl-substrate modification and mutations in the Ublconjugation machinery in the etiology and progression of a number of human diseases The primary aim of this review is to summarize the latest developments in our understanding of the different Ubl-protein modification systems, including the shared and unique features of these related pathways

Modification of Proteins by Ubiquitin and Ubiquitin-Like Proteins

Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc

https://science.sciencemag.org/content/sci/292/5526/2488.full.pdf

Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis

To succeed, many cells must alternate between life-styles that permit rapid growth in the presence of abundant nutrients and ones that enhance survival in the absence of those nutrients. One such change in life-style, the “acetate switch,” occurs as cells deplete their environment of acetate-producing carbon sources and begin to rely on their ability to scavenge for acetate. This review explains why, when, and how cells excrete or dissimilate acetate. The central components of the “switch” (phosphotransacetylase [PTA], acetate kinase [ACK], and AMP-forming acetyl coenzyme A synthetase [AMP-ACS]) and the behavior of cells that lack these components are introduced. Acetyl phosphate (acetyl∼P), the high-energy intermediate of acetate dissimilation, is discussed, and conditions that influence its intracellular concentration are described. Evidence is provided that acetyl∼P influences cellular processes from organelle biogenesis to cell cycle regulation and from biofilm development to pathogenesis. The merits of each mechanism proposed to explain the interaction of acetyl∼P with two-component signal transduction pathways are addressed. A short list of enzymes that generate acetyl∼P by PTA-ACKA-independent mechanisms is introduced and discussed briefly. Attention is then directed to the mechanisms used by cells to “flip the switch,” the induction and activation of the acetate-scavenging AMP-ACS. First, evidence is presented that nucleoid proteins orchestrate a progression of distinct nucleoprotein complexes to ensure proper transcription of its gene. Next, the way in which cells regulate AMP-ACS activity through reversible acetylation is described. Finally, the “acetate switch” as it exists in selected eubacteria, archaea, and eukaryotes, including humans, is described.

The acetate switch.

The sigma(S) (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little sigma(S), exposure to many different stress conditions results in rapid and strong sigma(S) induction. Consequently, transcription of numerous sigma(S)-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular sigma(S) level is achieved by rpoS transcriptional and translational control as well as by regulated sigma(S) proteolysis, with various stress conditions differentially affecting these levels of sigma(S) control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of sigma(S), which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of sigma(S) regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For sigma(S) proteolysis, the response regulator RssB is essential. RssB is a specific direct sigma(S) recognition factor, whose affinity for sigma(S) is modulated by phosphorylation of its receiver domain. RssB delivers sigma(S) to the ClpXP protease, where sigma(S) is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.

Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase.

Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.

Hfq and its constellation of RNA

The genome DNA of Escherichia coli is associated with about 10 DNA-binding structural proteins, altogether forming the nucleoid. The nucleoid proteins play some functional roles, besides their structural roles, in the global regulation of such essential DNA functions as replication, recombination, and transcription. Using a quantitative Western blot method, we have performed for the first time a systematic determination of the intracellular concentrations of 12 species of the nucleoid protein in E. coli W3110, including CbpA (curved DNA-binding protein A), CbpB (curved DNA-binding protein B, also known as Rob [right origin binding protein]), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Qβ), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td mutant phenotype A). Intracellular protein levels reach a maximum at the growing phase for nine proteins, CbpB (Rob), DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp, and StpA, which may play regulatory roles in DNA replication and/or transcription of the growth-related genes. In descending order, the level of accumulation, calculated in monomers, in growing E. coli cells is Fis, Hfq, HU, StpA, H-NS, IHF*, CbpB (Rob), Dps*, Lrp, DnaA, IciA, and CbpA* (stars represent the stationary-phase proteins). The order of abundance, in descending order, in the early stationary phase is Dps*, IHF*, HU, Hfq, H-NS, StpA, CbpB (Rob), DnaA, Lrp, IciA, CbpA, and Fis, while that in the late stationary phase is Dps*, IHF*, Hfq, HU, CbpA*, StpA, H-NS, CbpB (Rob), DnaA, Lrp, IciA, and Fis. Thus, the major protein components of the nucleoid change from Fis and HU in the growing phase to Dps in the stationary phase. The curved DNA-binding protein, CbpA, appears only in the late stationary phase. These changes in the composition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the genome functions.

Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid

The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases.

/pdf/development-of-an-elisa-based-on-the-baculovirus-expressed-16c61372y1.pdf

Development of an ELISA Based on the Baculovirus-Expressed Capsid Protein of Porcine Circovirus Type 2 as Antigen

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections

The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.

Isolation from Tobacco Mosaic Virus-Infected Tobacco of a Solubilized Template-Specific RNA-Dependent RNA Polymerase Containing a 126K/183K Protein Heterodimer

Prevalence of swine Torque teno virus genogroups 1 and 2 in Japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex.

Susumu Ueda

Papers

Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid

Development of an ELISA Based on the Baculovirus-Expressed Capsid Protein of Porcine Circovirus Type 2 as Antigen

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections

Isolation from Tobacco Mosaic Virus-Infected Tobacco of a Solubilized Template-Specific RNA-Dependent RNA Polymerase Containing a 126K/183K Protein Heterodimer

Prevalence of swine Torque teno virus genogroups 1 and 2 in Japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex.